Polynucleotide sequencing methods

ABSTRACT

Methods for determining the sequence of a polynucleotide comprising the steps of (i) contacting a polynucleotide processive enzyme immobilised in a fixed position, with a target polynucleotide under conditions sufficient to induce enzyme activity; (ii) detecting an effect consequent on the interaction of the enzyme and the polynucleotide, wherein the effect is detected by measurement of a non-linear optical signal or a linear signal coupled to a non-linear signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/478,036, filed Nov. 17, 2003, which is a National Stage Entry ofPCT/GB02/02345, filed May 20, 2002, which claims the benefit of GB0112238.1, filed May 18, 2001, the entire contents of which are eachincorporated herein by reference.

FIELD

This invention relates to a method for determining the sequence of apolynucleotide.

BACKGROUND

The ability to determine the sequence of a polynucleotide is of greatscientific importance, as shown by the Human Genome Project in mappingthe three billion bases of DNA encoded in the human genome.

The principal method in general use for large-scale DNA sequencing isthe chain termination method. This method was first developed by Sangerand Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977; 74:5463-5467), and relies on the use of dideoxy derivatives of the fournucleoside triphosphates which are incorporated into the nascentpolynucleotide chain in a polymerase reaction. Upon incorporation, thedideoxy derivatives terminate the polymerase reaction and the productsare then separated by gel electrophoresis and analysed to reveal theposition at which the particular dideoxy derivative was incorporatedinto the chain.

Although this method is widely used and produces reliable results, it isrecognised that it is slow, labour-intensive and expensive.

Fluorescent labels have been used to identify nucleotide incorporationonto a growing nascent DNA molecule, using the polymerase reaction (seeWO91/06678). However, these techniques have the disadvantage ofincreasing background interference from the fluorophores. As the DNAmolecule grows, the background “noise” increases and the time requiredto detect each nucleotide incorporation needs to be increased. Thisseverely restricts the use of the method for sequencing largepolynucleotides. The most serious limitation of polynucleotidesequencing systems built around fluorescent dyes, however, is theproblem of photobleaching.

Photobleaching is a well-documented phenomenon in fluorescent dyesystems and results from exposure of the dye to excitation wavelengths.All dye systems have an ability to absorb a limited number of photonsbefore photobleaching occurs. Once photobleaching has occurred thefluorescent dye is no longer visible to the observer and hence, ifconjugated to a molecule, this will not be detectable.

There is therefore a need for an improved method for determining thesequence of a polynucleotide, which significantly increases the rate andfragment size of the polynucleotide being sequenced and which preferablydoes not depend on fluorescently labelled nucleotides for detection.Further, the method should be capable of being carried out by anautomated process, reducing the complexity and cost associated withexisting methods.

SUMMARY

The present invention is based on the realisation that a conformationaland/or mass and/or energy distribution change in a polynucleotideprocessive enzyme, which occurs when an enzyme associates with and movesalong a target polynucleotide, can be detected using non-linear opticalimaging, including that based on second or third harmonic generation.

According to the present invention, a method for sequencing apolynucleotide comprises the steps of:

(i) contacting a polynucleotide processive enzyme, immobilised in afixed position, with a target polynucleotide under conditions sufficientfor enzyme activity; and

(ii) detecting an effect consequent on the interaction of the enzyme andthe polynucleotide, wherein the effect is detected by measurement of anon-linear optical signal or a linear signal coupled to a non-linearsignal.

Numerous advantages are achieved with the present invention. Sequencingcan be carried out with small amounts of polynucleotide, with thecapability of sequencing single polynucleotide molecules, therebyeliminating the need for amplification prior to initiation ofsequencing. Long sequence read lengths can be obtained and secondarystructure considerations minimised Obtaining long read lengthseliminates the need for extensive fragment reassembly using computation.Further, as the invention is not dependent upon the need forfluorescently-labelled nucleotides or any measurement of fluorescence,the limitation of read length at the single molecule level as a functionof photobleaching or other unpredictable fluorescence effects, iscircumvented. The present invention also permits long polynucleotidefragments to be read sequentially by the same enzyme system. This hasthe benefit of allowing a single enzyme system to be used which can beregenerated and re-used allowing many different polynucleotide templatesto be sequenced. Finally, the utilisation of Second or Third HarmonicGeneration offers advantages due to the lack of photodamage andphotobleaching. This is due to the fact that no photochemistry occurs,even in the focal plane because the signal, stimulated by non-resonantradiation, does not involve an excited state with a finite lifetime.

According to a second aspect of the invention, a solid support materialcomprises at least one polymerase and at least one dipolar moleculepositioned on or proximal to the polymerase.

According to a third aspect of the invention, an imaging system set upto detect a non-linear optical signal, comprises a solid support havingimmobilised thereon an enzyme that interacts with a polynucleotide, anda dipolar molecule positioned on or proximal to the enzyme.

DESCRIPTION OF THE DRAWINGS

The invention is described with reference to the accompanying figure,wherein:

FIG. 1 is a schematic illustration of an imaging system that utilisessecond harmonic generation; and

FIG. 2 shows the second harmonic signal generated by a polymerase onincorporation of a specific polynucleotide (SEQ ID NO: 3).

DETAILED DESCRIPTION

The present invention makes use of conventional non-linear opticalmeasurements to identify a conformational and/or mass and/or energydistribution change occurring as a polynucleotide processive enzymeinteracts with the individual bases on a target polynucleotide orincorporates nucleotides onto a nascent polynucleotide molecule.

The use of non-linear optical methods for imaging molecules is known.What has not been appreciated is that these methods can be applied tothe sequencing of a polynucleotide, making use of an immobilised orfixed enzyme.

In a separate embodiment, a linear signal is generated in addition to anon-linear signal and the linear signal is detected. The two signals aresaid to be coupled, resulting in enhanced detection.

The term “polynucleotide” as used herein is to be interpreted broadly,and includes DNA and RNA, including modified DNA and RNA, DNA/RNAhybrids, as well as other hybridising nucleic acid-like molecules, e.g.peptide nucleic acid (PNA).

The term “polynucleotide processive enzyme” as used herein is to beinterpreted broadly and relates to any enzyme that interacts with apolynucleotide and moves continuously along the polynucleotide. Theenzyme is preferably a polymerase enzyme, and may be of any known type.For example, the polymerase may be any DNA-dependent DNA polymerase. Ifthe target polynucleotide is a RNA molecule, then the polymerase may bea RNA-dependent DNA polymerase, i.e. reverse transcriptase, or aRNA-dependent RNA polymerase, i.e. RNA replicase. In a preferredembodiment of the invention, the polymerase is T4 polymerase. In furtherpreferred embodiments of the invention, the polymerase is either E. colipolymerase III holoenzyme (McHenry, Ann. Rev. Biochem., 1988; 57:519);T7 polymerase (Schwager et al., Methods in Molecular and CellularBiology, 1989/90; 1(4): 155-159) or bacteriophage T7 gene 5 polymerasecomplexed with E. coli Thioredoxin (Tabor et al., J. Biol. Chem., 1987;262: 1612-1623). Each of these polymerase enzymes binds to a targetpolynucleotide with high processivity (and fidelity) and thereforemaintains a polymerase-polynucleotide complex, even when polymerisationis not actively taking place.

Alternative enzymes that interact with a polynucleotide includehelicase, primase, holoenzyme, topoisomerase or gyrase enzymes. Suchenzymes offer further advantages. For example, using a helicase reducesthe problem of secondary structures that exist within polynucleotidemolecules, as helicases encounter and overcome these structures withintheir natural environment. Secondly, helicases allow the necessaryreactions to be carried out on double-stranded DNA at room temperature.

As the enzyme interacts with successive bases on the polynucleotide, itsconformation will change depending on which base (or nucleotide) on thetarget it is brought into contact with. Thus, the temporal order of basepair additions during the reaction is measured on a single molecule ofnucleic acid, i.e. the activity of the enzyme system on the templatepolynucleotide to be sequenced can be followed in real time. Thesequence is deduced by identifying which base (nucleotide) is beingincorporated into the growing complementary strand of the targetpolynucleotide via the catalytic activity of the enzyme.

An important aspect of the present invention is the immobilisation ofthe enzyme in a fixed position relative to the imaging system. This ispreferably carried out by immobilising the enzyme to a solid support,with the enzyme retaining its biological activity. Methods for theimmobilisation of suitable enzymes to a solid support are known. Forexample, WO-A-99/05315 describes the immobilisation of a polymeraseenzyme to a solid support. General methods for immobilising proteins tosupports are suitable.

The optical detection methods used in the present invention are intendedto image at the single molecule level, i.e. to generate a distinctimage/signal for one enzyme. A plurality of enzymes may be immobilisedon a solid support at a density that permits single enzyme resolution.Therefore, in one embodiment, there are multiple enzymes immobilised ona solid support, and the method of the invention can be carried out onthese simultaneously. This allows different polynucleotide molecules tobe sequenced together.

It will be apparent to the skilled person to carry out the imagingmethod under conditions suitable to promote enzymatic activity. Forexample, with regard to a polymerase enzyme, it will be apparent thatthe other components necessary for the polymerase reaction to proceed,are required. In this embodiment, a polynucleotide primer molecule andeach of the nucleoside triphosphates dATP, dTTP, dCTP and dGTP, will berequired. The nucleoside triphosphates may be added sequentially, withremoval of non-bound nucleotides prior to the introduction of the nextnucleoside triphosphate. Alternatively, all the triphosphates can bepresent at the same time. It may be preferable to utilise triphosphatesthat have one or more blocking groups which can be removed selectivelyby pulsed monochromatic light, thereby preventing non-controlledincorporation. Suitable blocked triphosphates are disclosed inWO-A-99/05315.

High-resolution non-linear optical imaging systems are known in the art.In general, the non-linear polarisation for a material can be expressedas:

P=X ⁽¹⁾ E ¹ +X ⁽²⁾ E ² +X ⁽³⁾ E ³+

where P is the induced polarisation, X^((n)) is the nth-order non-linearsusceptibility, and E is the electric field vector. The first termdescribes normal absorption and reflection of light; the seconddescribes second harmonic generation (SHG), sum and difference frequencygeneration; and the third describes light scattering, stimulated Ramanprocesses, third harmonic generation (TGH), and both two- andthree-photon absorption.

A preferred imaging system of the present invention relies on thedetection of the signal arising from second or third harmonicgeneration.

Single-molecule resolution using second or third harmonic generation(hereinafter referred to as SHG) is known in the art (Peleg et al.,Proc. Natl. Acad. Sci. USA, 1999; 95:6700-6704 and Peleg et al.,Bioimaging, 1996; 4:215-224).

The general set-up of the imaging system can be as described in Peleg etal., 1996, supra, and as shown in FIG. 1. With reference to FIG. 1, alaser (1) is used as the illumination source, to generate a laser beamwhich is then passed through a polarizer (2). Part of the laser beam maybe directed through a non-linear crystal (3) to produce a green beam toaid the alignment of the laser beam. A photodiode (4) is placed in closeproximity to the optical path in order to provide a means to monitor thegenerated near-infrared (NIR) intensity. A filter (5) is positioned infront of the entrance port of a microscope to prevent any secondharmonic from entering the microscope. The laser beam is focused ontothe solid support comprising the immobilised enzyme, and the non-linearsignal is collected by lenses (7) and detected using a monochromator(8). The fundamental intensity is blocked using an IR filter. The signalfrom the photomultiplier is amplified, averaged and integrated using aboxcar averager and channel integrator (9). The generated signals arethen transferred to a computer (10) to generate the images.

In order to generate the second or third harmonic, it is necessary toposition an appropriate label on or in close proximity to theimmobilised enzyme. Highly dipolar molecules are suitable for thispurpose. (Lewis et al. Chem. Phys., 1999; 245:133-144). An example ofsuitable molecules are dyes, particularly styryl dyes (such as membranedye JPW 1259—supplied by Molecular Probes). Green Fluorescent Protein(GFP) is another example of a “dye” or “label” which can be used toimage via SHG. As used herein, GFP refers to both the wild-type protein,and spectrally shifted mutants thereof (Tsien, Ann. Rev. Biochem., 1998;67:509 and U.S. Pat. No. 5,777,079 and U.S. Pat. No. 5,625,048). Othersuitable dyes include di-4-ANEPPS, di-8-ANEPPS and JPW2080 (MolecularProbes).

The dipolar molecules may be located on the individual bases of thepolynucleotide (or its complement if the dipolar molecules are attachedto the nucleoside triphosphates and used in a polymerase reaction).

In a preferred embodiment of the invention, the enzyme, e.g. apolymerase, is prepared as a recombinant fusion with GFP. The GFP can belocated at the N- or C-terminus of the enzyme (the C-terminus may bedesirable if a polymerase is to be used in conjunction with a “slidingclamp”). Alternatively, the GFP molecule can be located anywhere withinthe enzyme, provided that enzymatic activity is retained.

In a separate embodiment of the present invention, the non-linearoptical imaging system is Raman spectroscopy or surface enhanced Ramanspectroscopy (SERS). An overview of Raman spectroscopy is contained inMcGilp, Progress in Surface Science, 1995; 49(1):1-106.

The optical radiation used to excite the Raman system is, preferably,Near Infrared Radiation (NIR). NIR excitation has the advantage ofdecreasing the fluorescence and Raman signal of the surrounding mediumor solvent.

In a separate embodiment of the invention, the non-linear signal can beenhanced by the use of a metal nanoparticle and/or a roughened metalsurface (Boyed et al., Phys Rev., 1984; B. 30:519-526, Chen et al.,Phys. Rev. Lett., 1981; 46:1010-1012 and Peleg et al., 1996, supra). Asignal enhancing metal nanoparticle can be conjugated to the enzyme(e.g. with a nanoparticle conjugated antibody, Lewis et al., Proc. Natl.Acad. Sci. USA, 1999; 96:6700-6704), immobilised near theimmobilised/localised enzyme or brought into close proximity to the SHGdye/enzyme.

A metal nanoparticle enhances the spectroscopic imaging associated with,in particular, SHG from nanometric regions, thereby permitting improvedimaging at the single molecule level. Spectroscopic imaging based onRaman scattering can also be improved using a metal nanoparticle.Suitable metal nanoparticles are known, and include gold and silvernanoparticles. The nanoparticles are generally of a diameter of from 5nm to 100 nm, preferably from 10 nm to 60 nm. The nanoparticles can beattached to the polynucleotide (or its complement if the nanoparticlesare attached to nucleoside triphosphates and used in a polymerasereaction).

A roughened metal surface has also been shown to improve the sensitivityof the SHG process (Chen et al., 1981, supra and Peleg et al., 1996,supra) and is also a requirement for SERS. The metal surface is usuallysilver or another noble metal. An initial selective modification of themetal surface at sub-wavelength spatial resolution can be carried outusing various techniques, including the use of atomic force microscopy(AFM). A platinum-coated AFM tip can be used to catalyse hydrogenationof terminal azides to amino groups that are amenable to furtherderivatisation (Muller et al., Science, 1995; 268:272-273). The enzymescan then be placed into “hot spots” where high local fields exist inregions where optical modes are localised (Shalaev et al. Phys. Rep.,1996; 272:61).

In a separate embodiment of the invention, a nanoparticle can be broughtinto close proximity with the enzyme using an AFM cantilever tip/probe,to thereby enhance the non-linear signal.

AFM has been shown recently to be capable of having a time resolutionand sensitivity applicable to the dynamic imaging of proteinconformational changes (Rousso et al., J. Struc. Biol., 1997;119:158-164). This is utilised in a preferred embodiment of theinvention, where an AFM probe/tip is positioned over the enzyme and, incombination with non-linear optical information (e.g. SHG), used todetect conformational changes of a protein due to the interactionbetween the enzyme and the nucleotide sequence as the enzyme moves alongthe target polynucleotide. The information may be collected in thefar-field using conventional confocal optics or in reflection mode ifused in conjunction with total internal reflection.

In a further embodiment, the non-linear signal (e.g. SHG) is monitoredin the near-field using Near-Field Scanning Optical Microscopy (NSOM).NSOM is a form of scanning probe microscopy, which makes use of theoptical interaction between a nanoscopic tip (as used in AFM) and asample to obtain spatially resolved optical information. Near-fieldmicroscopy in combination with SHG has been studied extensively andshown to be surface sensitive on an atomic scale (McGilp, 1995, supra).The main advantage of using NSOM as part of the imaging system is thatit allows a large increase in resolution to sub-wave-length dimensions.As the present invention relates to the conformational monitoring of asingle enzyme, e.g. a polymerase enzyme, as it interacts with apolynucleotide, sub-wave-length spatial resolution is highly desirable.In the context of this aspect of the invention, it is preferable if anAFM cantilever tip is used as an apertureless Near-field scanningmicroscope (Sangohdar et al, J. Opt. A: Pure Appl. Opt., 1999; 523-530).This is analogous to the use of metallic nanoparticles as a source oflocal field enhancement. It is preferred that the tip is made out of, orcoated with, a noble metal or any material which acts to increase thelocal electromagnetic field. Alternatively, a metallic nanoparticle maybe connected directly to the cantilever tip. This has already been shownto be applicable to the monitoring of conformational changes at thesingle molecule level (Rousso, et al. supra).

In a further separate embodiment of the present invention, anindependently generated surface plasmon (or polariton)/evanescent fieldcan be used to enhance the signal-to-noise ratio of the non-linearsignal. This evanescent wave enhanced imaging technique has greatersignal-to-noise ratio than, for example, SHG imaging alone. In thisembodiment, the evanescently enhanced SHG field signal from the labelledenzyme can be collected in the near field by an NSOM fibre whilstsimultaneously obtaining AFM conformational data, and at the same timethe amount of absorbed evanescent radiation can be monitored to obtaininformation on the amount of coupling between the evanescent field andthe labelled polymerase/SHG field.

In this configuration (NSOM collection mode) the system acts as a photonscanning tunneling microscope (PSTM) and the evanescent or surfaceplasmon field is coupled into the NSOM fibre probe tip. Any attenuationin the field strength of the signal reaching the tip by the polymerasewill be monitored via a detector positioned at the end of the tip.

Surface plasmon resonance is known in the art, and relies on thegeneration of an evanescent wave by applying an incident light beam to aprism. A typical set-up for use in this embodiment consists of a prismwhich is coupled optically to a metal coated glass coverslip on which anenzyme is immobilised. The coverslip is part of a microfluidic flow cellsystem with an inlet for introducing ligands (nucleotides) over theimmobilised enzyme. The enzyme is also labelled to allow non-lineareffects to be generated. An incident light beam is applied to the prismto generate the surface plasmon field. At the same time, a non-linearsignal (e.g. second harmonic field) is generated by directing a pulsednear infrared laser through a polarizer and half wave plate, into anoptical scanner for beam control via a filter to eliminate opticalsecond harmonic noise, and then into the sample. The non-linear opticalsignal is collected with lenses and a filter and directed into amonochromator, passed to a photomultiplier tube for detection and thenamplified and recorded via a computer system.

When the non-linear optical is coupled to that generating the evanescentfield, the signal that is detected can also be the linear (evanescent)signal. In this embodiment, the NSOM can be used in the collection madeto detect the linear signal.

In a separate aspect of the present invention, the polynucleotidesequencing can be carried out within a cell.

It has been demonstrated that, in its native cellular environment, a DNApolymerase and its associated replisome complex is anchored in place (orlocalised in space) within the cell (Newport et al., Curr. Opin. CellBiol., 1996; 8:365; and Lemon et al., Science, 1998; 282:1516-1519. Thisnative anchored replication complex is analogous to the immobilisationof the enzyme to a solid support.

This allows the in vivo monitoring of conformational and templatesequence-related changes of replisome-related molecules at the singlemolecule level to be carried out in real-time during DNA replicationand/or cell division.

In order to carry out this aspect, it is necessary to modify the enzymeso that it can be imaged using nonlinear optical detection techniques.This can be achieved by genetic fusion of the enzyme with, for example,green fluorescent protein (GFP). The cell should also be immobilised topermit detection to occur.

The expressed fusion protein can be monitored/detected at its anchoredcellular location via the application of non-linear optical detection(second harmonic generation).

The following Example illustrates the invention.

In this experiment, a fusion protein of Green Fluorescent Protein (GFP)and a polymerase was created via recombinant techniques well known inthe art.

Quartz chips (14 mm in diameter, 0.3 mm thick) were spin-coated with a50 nm thick layer of gold and then coated with a layer of planardextran. These gold coated quartz chips were then placed into the fluidcell of a custom built Nearfield Scanning Optical Microscope (NSOM). Thegold-coated quartz chips were coupled optically to a quartz prism viaindex matching oil. The fluid cell was then sealed and polymerase bufferwas then allowed to flow over the chip.

Immobilisation of the polymerase to the chip surface was carried outaccording to Jonsson et al., Biotechniques, 1991; 11:620-627. The chipenvironment was equilibrated with running buffer (10 mM hepes, 10 mMMgCl₂ 150 mM NaCl, 0.05% surfactant P20, pH 7.4). Equal volumes ofN-hydroxysuccinimide (0.1 M in water) andN-ethyl-N′-(dimethylaminopropyl) carbodiimide (EDC) (0.1 M in water)were mixed together and injected across the chip surface, to activatethe carboxymethylated dextran. The polymerase-GFP fusion protein (150μl) was mixed with 10 mM sodium acetate (100 μl, pH 5) and injectedacross the activated surface. Finally, residual N-hydroxysuccinimideesters on the chip surface were reacted with ethanolamine (35 μl, 1 M inwater, pH 8.5), and non-bound polymerase was washed from the surface.The immobilization procedure was performed with a continuous flow ofrunning buffer (5 μl/min) at a temperature of 25° C.

50 μl of antibody binding buffer (10 mM MES pH 6.0, 150 mM NaCl, 3 mMEDTA) was flowed over the immobilized polymerase/GFP on the chip surfaceat a flow rate of 5 μl/min at 25° C. A primary antibody (GFP (B-2)Bbiotin conjugated 200 μl ml-1, Santa Cruz Biotechnology) was diluted1:3000 in antibody binding buffer and allowed to flow over the chipsurface at a flow rate of 5 μl/min for 30 minutes. Excess antibody wasthen washed off the surface by flowing antibody binding buffer over thechip at a flow rate of 5 μl/min for 30 minutes.

A secondary antibody (Immunogold conjugate EM Goat antimouse IgG (H+L)40 nm, British Biocell International) was diluted 1:1000 in antibodybinding buffer and allowed to flow over the chip surface at a flow rateof 5 μl/min for 30 minutes. Excess antibody was then washed off thesurface by flowing antibody binding buffer over the chip at a flow rateof 5 μl/min for 30 minutes. The buffer was then returned to runningbuffer which was then allowed to flow over the chip at a rate of 5μl/min for 30 minutes before initiation of the next stage.

Two oligonucleotides were synthesized using standard phosphoramiditechemistry. The oligonucleotide defined as SEQ ID NO. 1 was used as thetarget polynucleotide, and the oligonucleotide defined as SEQ ID NO. 2was used as the primer.

SEQ ID NO: 1 CAAGGAGAGGACGCTGCTTGTCGAAGGTAAGGAACGGACGAGAG AAGGGAGAGSEQ ID NO: 2 CTCTCCCTTCTCTCGTC

The two oligonucleotides were reacted under hybridizing conditions toform the target-primer complex. The primed DNA was then suspended inbuffer (20 mM Tris-HCl, pH 7.5, 8 mM MgCl₂, 4% (v/v) glycerol, 5 mMdithiothreitol (DDT) containing 150 μl of the β sub-units that form asliding-clamp complex around the primer DNA. This process is known aspre-initiation.

In order to detect the conformational changes in the polymerase, amodified NSOM was used in tapping mode, with pulled quartz multimode 100μm long fibre cantilevers. The cantilever was driven close to itsresonant frequency and an initial area scan was carried out over thesurface of the chip containing immobilized antibodies. The secondharmonic signal was generated from the immobilized polymerase in theflow cell via initial illumination from a pulsed Near infra-red lasersource. The NSOM tip was then scanned over the chip surface in the flowcell in order to obtain an image of a 40 nm gold particles in the flowcell which is associated with the polymerase. The tip is then held instationary mode over the polymerase.

The pre-initiated pre-primed complex was then injected into the flowcell at a flow rate of 5 μl/min so that the “clamp” around theprimer-template molecule forms a complex with the immobilizedpolymerase. The flow cell was maintained at 25° C. by a cooling devicebuilt into the flow cell.

The running buffer was then flushed continuously through the flowcell at500 μl/min. After 10 minutes the sequencing reaction was initiated byinjection of 0.4 mM dATP (8 μl) into the buffer at a flow rate of 500μl/min. After 4 minutes 0.4 mM dTTP (8 μl) was injected into theflowcell. Then after another 4 minutes 0.4 mM dGTP (8 μl) was injectedand after another 4 minutes 0.4 mM dCTP (8 μl) was injected. This cyclewas then repeated 10 times. Over the entire time period the secondharmonic signal transmitted via the multimode fibre was passed into amonochromator and then into a photomultiplier. The signal from thephotomultipler was then amplified and fed into a computer for processingand storage.

The intensity change of second harmonic signal arising from thepolymerase complex for a period of 10 seconds from the start of eachinjection was then calculated and plotted against nucleotide injectedinto the flow cell.

The results of the sequencing reaction are shown in FIG. 2. As can beseen from the graph, large intensity changes (larger intensity changesaccounting for identical nucleotides adjacent to each other) correspondto the complement of that of SEQ ID NO. 1 (reading from right to left,minus that part of which hybridizes to the primer sequence).

1. A method for determining the sequence of a polynucleotide, comprisingthe steps of: (i) contacting a polynucleotide processive enzymeimmobilised in a fixed position, with a target polynucleotide underconditions sufficient to induce enzyme activity, wherein a dipolarmolecule is positioned on or proximal to the enzyme, and wherein theenzyme activity occurs in the presence of the nucleoside triphosphatesdATP, dTTP, dGTP, dCTP, or combinations thereof; (ii) detecting aneffect consequent on the interaction of the enzyme and polynucleotide,therein determining the sequence of the target polynucleotide; whereinthe dipolar molecule is attached to the individual bases of the targetpolynucleotide, wherein the effect is detected by measurement of anon-linear optical signal or a linear signal coupled to a non-linearsignal, and wherein the detection method comprises Near-Field ScanningOptical Microscopy.
 2. The method according to claim 1, wherein theeffect is detected by measurement of a non-linear signal.
 3. The methodaccording to claim 1, wherein the non-linear optical detection is secondor third harmonic generation imaging.
 4. The method according to claim1, wherein the detection method further comprises Raman spectroscopy orsurface enhanced Raman spectroscopy.
 5. The method according to claim 1,wherein the dipolar molecule is a styryl dye molecule.
 6. The methodaccording to claim 1, wherein the dipolar molecule is green fluorescentprotein.
 7. The method according to claim 1, wherein the enzyme is apolymerase.
 8. The method according to claim 1, wherein the enzyme is ahelicase or primase enzyme.
 9. The method according to claim 1, whereinthe enzyme activity occurs in the presence of the nucleosidetriphosphates dATP, dTTP, dGTP and dCTP.
 10. The method according toclaim 9, wherein the nucleoside triphosphates comprise one or moreblocking groups which can be removed selectively by pulsed monochromaticlight.
 11. The method according to claim 1, wherein a metal nanoparticleis positioned on or proximal to the enzyme.
 12. The method according toclaim 11, wherein the nanoparticle is a gold or silver nanoparticle. 13.The method according to claim 11, wherein the nanoparticle isincorporated onto one or more of the individual bases of thepolynucleotide.
 14. The method according to claim 1, wherein the enzymeis immobilised on a solid support.
 15. The method according to claim 14,wherein there are a plurality of enzymes immobilised on the solidsupport.
 16. The method according to claim 14, wherein the solid supporthas a roughened metal surface.
 17. The method according to claim 14,wherein the support is silver or gold.
 18. The method according to claim1, further comprising the application of localized surface plasmonresonance.
 19. The method according to claim 1, wherein the enzyme isimmobilised in a fixed position within a cell.
 20. The method accordingto claim 1, wherein the nucleoside triphosphates comprise one or moreblocking groups.
 21. A method for determining the sequence of apolynucleotide, comprising the steps of: (i) contacting a polynucleotideprocessive enzyme immobilised in a fixed position, with a targetpolynucleotide under conditions sufficient to induce enzyme activity,wherein a dipolar molecule is positioned on or proximal to the enzyme,and wherein the enzyme activity occurs in the presence of the nucleosidetriphosphates dATP, dTTP, dGTP, dCTP, or combinations thereof; (ii)detecting an effect consequent on the interaction of the enzyme andpolynucleotide, therein determining the sequence of the targetpolynucleotide; wherein a metal nanoparticle is positioned on orproximal to the enzyme, wherein the nanoparticle is a gold or silvernanoparticle and wherein the nanoparticle is incorporated onto one ormore of the individual bases of the polynucleotide; wherein the effectis detected by measurement of a non-linear optical signal or a linearsignal coupled to a non-linear signal, and wherein the detection methodcomprises Near-Field Scanning Optical Microscopy.
 22. A method fordetermining the sequence of a polynucleotide, comprising the steps of:(i) contacting a polynucleotide processive enzyme immobilised in a fixedposition, with a target polynucleotide under conditions sufficient toinduce enzyme activity, wherein a dipolar molecule is positioned on orproximal to the enzyme, and wherein the enzyme activity occurs in thepresence of the nucleoside triphosphates dATP, dTTP, dGTP, dCTP, orcombinations thereof; (ii) detecting an effect consequent on theinteraction of the enzyme and polynucleotide, therein determining thesequence of the target polynucleotide; wherein the enzyme is immobilisedin a fixed position within a cell, wherein the effect is detected bymeasurement of a non-linear optical signal or a linear signal coupled toa non-linear signal, and wherein the detection method comprisesNear-Field Scanning Optical Microscopy.